[go: up one dir, main page]

US20060141461A1 - Methods of assessing the risk of reproductive failure by measuring telomere length - Google Patents

Methods of assessing the risk of reproductive failure by measuring telomere length Download PDF

Info

Publication number
US20060141461A1
US20060141461A1 US10/531,964 US53196405A US2006141461A1 US 20060141461 A1 US20060141461 A1 US 20060141461A1 US 53196405 A US53196405 A US 53196405A US 2006141461 A1 US2006141461 A1 US 2006141461A1
Authority
US
United States
Prior art keywords
telomere
chromosome
oocyte
length
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/531,964
Other languages
English (en)
Inventor
David Keefe
Original Assignee
Women and Infants Hospital of Rhode Island
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Assigned to WOMEN AND INFANTS HOSPITAL OF RI reassignment WOMEN AND INFANTS HOSPITAL OF RI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KEEFE, DAVID
Application filed by Women and Infants Hospital of Rhode Island filed Critical Women and Infants Hospital of Rhode Island
Priority to US10/531,964 priority Critical patent/US20060141461A1/en
Publication of US20060141461A1 publication Critical patent/US20060141461A1/en
Priority to US12/482,176 priority patent/US8771941B2/en
Assigned to KEEFE, DAVID L. reassignment KEEFE, DAVID L. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WOMEN AND INFANTS HOSPITAL OF RHODE ISLAND, INC.
Priority to US14/323,638 priority patent/US20150167081A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/42—Gynaecological or obstetrical instruments or methods
    • A61B17/425—Gynaecological or obstetrical instruments or methods for reproduction or fertilisation
    • A61B17/435—Gynaecological or obstetrical instruments or methods for reproduction or fertilisation for embryo or ova transplantation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Telomeres are repeated sequences of DNA that cap the ends of chromosomes and prevent the formation of end-to-end fusions. During normal DNA replication, the ends of chromosomes, or telomeres, are left unreplicated. This results in the loss of a small amount of DNA from each chromosome with every cell cycle. This loss is subsequently corrected by an enzyme known as telomerase. Telomerase is a cellular enzyme, which is directed to the nucleotide polymerization or maintenance of telomeres, and contains a complex of protein components and an integral RNA component.
  • Vertebrate telomeres consist of tandem repeats of the sequence TTAGGG and associated proteins, which cap the ends of chromosomes and protect them from degradation and fusion (Blackburn E. H., Cell 106: 661-673 , Nature 408(6808): 53-6. 2001). Extensive evidence has shown that telomere shortening and dysfunction in cultured somatic cells leads to so-called replicative senescence (Blackburn E. H., Nature 408(6808): 53-6, 2000). In turn, reversal of telomere shortening by forced expression of telomerase rescues cells from senescence and extends cell life span indefinitely (Bodnar, A. G., M. Ouellette, et al., Science 279(5349): 349-52, 1998). (Vaziri and Benchimol et al., Curr Biol 8(5): 279-82. 1998).
  • Telomeres cap and protect the ends of chromosomes, establish homologue pairing and enhance chiasmata formation during early meiosis, and shorten with cell division and exposure to reactive oxygen (ROS) to mediate cellular aging in somatic cells.
  • Germ cells like stem cells and cancer cells, contain telomerase, a reverse transcriptase which maintains telomeres by adding telomere repeats to the 3 prime end of DNA, thus allowing germ cells to largely bypass the senescence response exhibited by cells with critically short telomeres.
  • telomere elongation is variable and stochastic in most cell types, and in females, telomerase activity decreases during late meiosis, so telomere length, determined during early development, provides a developmental bottleneck. Germ cells with adequate telomere length pass across generations, but the fate of eggs “stuck in the bottleneck” by short telomeres is poorly understood.
  • telomere reverse transcriptase is the primary enzyme for maintaining the length of telomere repeats.
  • Telomerase activity is present during early oogenesis, but is almost absent during late oogenesis and early preimplantation embryo development until the morula stage of development.
  • telomere length in oocytes and early embryos is established during early development.
  • telomeres reach a critically short length, the cell undergoes cell cycle arrest and apoptosis, so late oogenesis and early preimplantation embryo development may represent a kind of bottleneck for telomere length during development.
  • RNA component of the human enzyme contains a short region complementary to the human telomeric repeat sequence (Feng et al. (1995) Science, 269:1236). Somatic cells lack telomerase activity, and their telomeres have been found to shorten with cell division both in vivo and in culture. In germ cells and embryos, telomerase actively restores telomeres, so that the chromosomes do not shorten progressively across generations. The short and numerous cell cycles of replicating primordial germ cells and oogonia during oogenesis, however, challenge telomerase to keep up with the progressive telomere shortening. In mature oocytes and early stage pre-implantation embryos, telomerase is down regulated until the blastocyst stage of development.
  • telomere length of chromosomes of an oocyte at fertilization can determine the resultant telomere length of the embryo.
  • Oocytes exhibit significant variability in telomere length. Oogonia exit mitosis after variable numbers of cell cycles, making the process of telomere shortening stochastic. This inevitable variability in telomere length of chromosomes from oocytes and embryos provides a cytogenetic mechanism to explain the most widely accepted theory of chromosomal aberration or aneuploidy in mammals—the production line hypothesis.
  • telomere shortening The telomeres of oocytes from late-exiting oogonia (late-ovulating oocytes) would be expected to be shorter than those of oocytes from early-exiting oogonia (early-ovulating oocytes).
  • GV germinal vesicle
  • chromosome pairing during leptotene/zygotense is promoted by a process of telomere clustering at the nuclear envelope, called bouquet formation, as chromosomes tethered at their telomeric ends find their homologous partner based on their similar sizes.
  • mice With increasing maternal age in women, meiotic chromosomes increasingly missegregate in human females, leading to aneuploidy, failed implantation, miscarriage, and increased rates of aneuploid offspring (Hassold, T., M. Abruzzo, et al,. Environ. Mol. Mutagen. 28(3): 167-75 (1996)).
  • checkpoints for meiotic chromosome behavior at metaphase-to anaphase transition are less efficient in females compared to males (Hunt, P., R. LeMaire, et al., Hum Mol Genet 4(11): 2007-12 (1995); LeMaire-Adkins, R., K. Radke, et al., J. Cell. Biol. 139(7): 1611-9 (1997)).
  • TR ⁇ / ⁇ mice which are deficient for the telomerase RNA and lack telomerase activity, show progressive telomere shortening with increasing mouse generations, eventually resulting in telomere-exhausted chromosomes and chromosomal end-to-end fusions (Blasco et al. (1997) Cell, 91:25-34; Gonzalez-Suarez et al (2000) Nat. Genetic. 26:114-117; Herrera et al. (1999a) EMBO 18:1172-1181; Herrera et al. (1999b) EMBO, 18:2950-2960; Lee et al. (1998) Nature, 392:569-574; Rudolph et al.
  • TR ⁇ / ⁇ mice Telomerase deficiency in TR ⁇ / ⁇ mice leads to the disruption of functional meiotic spindles and the misalignment of chromosomes during meiotic division of oocytes in late generation mice. In early generations, however, oocytes from TR ⁇ / ⁇ mice show no appreciable telomere dysfunction, and exhibit normal chromosome alignment during metaphase (Liu et al. (2002) Biol. Reprod. 64:204-210). Telomere dysfunction in late generation TR ⁇ / ⁇ mice leads to various pathologies including defects in development, growth, and immune function, as well as influences tumorigenesis. Female fertility also decreases with increasing TR ⁇ / ⁇ mouse generations, as evidenced by reduction in litter size, and compromised embryo development, eventually resulting in sterility (Herrera et al., 1999b; Lee et al., 1998).
  • Oocyte dysfunction is a major source of infertility and failed treatment in infertile women, even when the egg and embryo morphology appear normal.
  • Chromosomal abnormalities are the leading cause of oocyte dysfunction in aging women.
  • Aneuploidy trisomy and monosomy
  • the aberrant segregation of chromosomes during meiosis is the most commonly identified chromosomal abnormality in humans, observed in at least 35% of first trimester miscarriages, 4% of stillbirths and 0.3% of live-borns (Hassold, et al. (2001) Nat Rev Genet. 2(4):280-91).
  • Chromosomal aneuploidy is associated with a large number of genetic disorders that could be prevented or prepared for by appropriate diagnosis, e.g., Verp et al. (1990) Chap. 7, in Filkins and Russo, Eds., Human Prenatal Diagnosis.
  • Such disorders include Down's syndrome associated with chromosome 21 trisomy, Edward's syndrome associated with chromosome 18 trisomy, Plateau's syndrome associated with chromosome 13 trisomy, Tumer's syndrome associated with an absence of an X chromosome (XO), Kleinfelter's syndrome associated with an extra X chromosome (XXY), XYY syndrome, triple X syndrome, and the like.
  • IVF in vitro fertilization
  • FISH fluorescent in situ hybridization
  • the invention is based in part on the application of the quantitative fluorescent in situ hybridization (Q-FISH) assay to measure telomere length in oocytes and polar bodies from women as a means to predict risk of reproductive failure and aneuploidy in embryos and/or offspring, as well as to predict meitotic spindle morphology in oocytes and polar bodies.
  • Q-FISH quantitative fluorescent in situ hybridization
  • telomere length can be easily used as an indicator of reproductive failure and/or aneuploidy.
  • the invention provides a method for determining the risk of reproductive failure in a cell comprising:
  • the invention provides a method for determining the risk of reproductive failure in an oocyte comprising:
  • the invention provides a method for determining the risk of reproductive failure in a subject comprising:
  • the invention provides a method for determining the risk of reproductive failure in an oocyte comprising:
  • the invention also provides a method for determining the predisposition of an oocyte to reproductive failure comprising:
  • the invention features a method for selecting a fertilized oocyte with a low risk of reproductive failure for in vitro fertilization, comprising:
  • the invention provides a method of in vitro fertilization comprising:
  • the invention provides a method for optimizing the viability of an embryo comprising:
  • the invention also features a method for determining the risk of aneuploidy in a cell comprising:
  • a method for determining the risk of aneuploidy in a cell comprising:
  • the invention provides a method for determining the risk of aneuploidy in an oocyte comprising:
  • the invention provides a method for determining the risk of aneuploidy in an oocyte comprising:
  • the invention also features a method for selecting a fertilized oocyte with a low risk of aneuploidy for in vitro fertilization, comprising:
  • a related aspect of the invention is a method for determining the predisposition of an oocyte to aneuploidy comprising:
  • the invention also features a method of pre-implantation genetic testing to identify an oocyte with a predisposition to aneuploidy comprising:
  • the invention provides a kit for determining the risk of reproductive failure and/or aneuploidy in a cell comprising reagents for preparing a chromosomal spread from the cell or at least one cell in a population of cells representative of said cell; labeled telomere-specific repeat probes; reagents for performing quantitative fluorescent in situ hybridization (Q-FISH) analysis on the chromosomal spread; and instructions for measuring the length of a telomere obtained from the chromosomal spread, or obtained from a chromosome of said cell, and comparing the measured length of the telomere to the standardized average length of a control.
  • Q-FISH quantitative fluorescent in situ hybridization
  • FIG. 1 is a graph showing telomeric lengths from subjects at risk for reproductive failure. Eggs from unsuccessful cycles have shorter telomeres
  • FIG. 2 is a plot showing telomere lengths were highly correlated between eggs and polar bodies.
  • aneuploidy is intended to mean a condition wherein a cell contains an abnormal number of chromosomes. The term also encompasses the condition wherein individual genes are present in abnormal quantity, or wherein fragments of individual genes are present in abnormal quantity.
  • An abnormal number of chromosomes is a number greater than or less than the normal diploid number. In humans, aneuploidy is defined as any deviation from the normal human diploid number of 46 chromosomes.
  • the term “cell” is intended to include any eukaryotic cell, such as a mammalian somatic or germ line cell.
  • the cell of the invention contains DNA, which includes telomeres.
  • the cell is obtained from a human.
  • the cell is an oocyte.
  • a cell is from a population of cells and is representative of the cells in that population.
  • the cell of the invention is a somatic cell.
  • chromosome is intended to encompass a long structure composed of DNA and associated proteins. Chromosomes consist of nucleic acids. Each animal species has a defined number of chromosomes found in individual cells, referred to as the diploid number. The diploid number refers to the two copies of each homologous chromosome. Egg and sperm cells contain haploid numbers of chromosomes, or one copy of each homologous chromosome. The diploid number is restored at fertilization with the union of the sperm and egg.
  • chromosomal material is intended to include any number of chromosomes, or portion thereof, taken from an organism. In one embodiment of the invention, chromosomes are from an oocyte. In another embodiment, chromosomes of the invention are from the polar body of a fertilized or unfertilized egg.
  • embryo includes any animal in the early stages of growth and development following fertilization up to and including the blastocyst stage.
  • An embryo is characterized as having totipotent cells that are nondifferentiated.
  • somatic cells of an individual are cells of the body that are differentiated and not totipotent.
  • a “pre-implantation embryo” refers to a fertilized oocyte with two pronuclei (up to and including a blastocyst), which is not yet implanted in the lining of the female reproductive tract.
  • the pre-implantation embryo contains between about 2 and about 8 cells, although these ranges may vary among species.
  • the quality assessment for a human or a mouse embryo is performed on an embryo comprising between 2 and 8 cells (i.e., the embryo is assessed between about 18 and about 24 hours post-fertilization).
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as for example, phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • homologous nucleic acid sequences when compared, exhibit significant sequence identity or similarity.
  • the standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions. The hybridization conditions are described in greater detail below.
  • nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least 56%, more generally at least 59%, ordinarily at least 62%, more ordinarily at least 65%, often at least 68%, more often at least 71%, typically at least 74%, more typically at least 77%, usually at least 80%, more usually at least about 85%, preferably at least about 90%, more preferably at least about 95 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides.
  • substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence or fragment derived from any one of SEQ ID NOS: 1 through 10.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See Kanehisa (1984) Nuc. Acids Res. 12:203-213.
  • Percent identity and similarity between two sequences can be determined using a mathematical algorithm (see, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).
  • fluorescent in situ hybridization is intended to mean a nucleic acid hybridization technique which employs a fluorophor-labeled probe to specifically hybridize to, and thereby facilitate visualization of, a target nucleic acid.
  • in situ hybridization including FISH, is useful for determining the distribution of a nucleic acid in a nucleic acid-containing sample such as is contained in, for example, tissues at the single cell level. Such techniques have been used for karyotyping applications, as well as for detecting the presence, absence and/or arrangement of specific genes contained in a cell.
  • FISH involves the use of nucleic acid probes to determine if a particular nucleotide sequence is present in the chromosomal DNA of particular cells.
  • staining or “painting” are used interchangeably, and include hybridizing a probe to a chromosome or segment thereof, such that the probe reliably binds to the targeted chromosomal material therein and the bound probe is capable of being visualized.
  • Such methods are well known to those of ordinary skill in the art and are disclosed, for example, in U.S. Pat. No. 5,225,326; U.S. patent application Ser. No. 07/668,751; and PCT WO 94/02646, the entire contents of which are incorporated herein by reference.
  • the oligonucleotide probe in FISH is labeled with a fluorophor (fluorescent “tag” or “label”) according to standard practice.
  • the fluorophor can be directly attached to the probe (i.e., a covalent bond) or indirectly attached thereto (e.g., biotin can be attached to the probe and the fluorophor can be covalently attached to avidin; the biotin-labeled probe and the fluorophor-labeled avidin can form a complex which can function as the fluorophor-labeled probe in the method of the invention).
  • Fluorophors that can be used in accordance with the method of the invention are well known to those of ordinary skill in the art.
  • DAPI 4,6-diamidino-2-phenylindole
  • FITC fluorescein isothiocyanate
  • rhodamine see, for example, U.S. Pat. No. 4,373,932, for a list of exemplary fluorophors that can be used in accordance with the methods of the invention.
  • DAPI 4,6-diamidino-2-phenylindole
  • FITC fluorescein isothiocyanate
  • rhodamine see, for example, U.S. Pat. No. 4,373,932, for a list of exemplary fluorophors that can be used in accordance with the methods of the invention.
  • the existence of fluorophors having different excitation and emission spectrums from one another permits the simultaneous visualization of more than one target nucleic acid in a single fixed sample.
  • hybridization includes the process by which two complementary nucleic acid molecules anneal to one another.
  • Hybridization is a general technique in which the complementary strands of deoxyribonucleic acid (hereinafter “DNA”) molecules, ribonucleic acid (hereinafter “RNA”) molecules, and combinations of DNA and RNA are separated into single strands and then allowed to renature or reanneal and reform base-paired double helices.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • in situ hybridization is used which makes possible the detection and localization of specific nucleic acid sequences directly within an intact cell or tissue without any extraction of nucleic acids whatsoever.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other.
  • Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology , Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual , Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11.
  • in situ hybridization is intended to mean a nucleic acid hybridization technique which employs a labeled probe to specifically hybridize to and thereby, facilitate visualization of, a target nucleic acid.
  • In situ hybridization is performed by denaturing the target nucleic acid so that it is capable of hybridizing to a complementary probe contained in a hybridization solution.
  • the fixed sample may be concurrently or sequentially contacted with the denaturant and the hybridization solution.
  • the fixed sample is contacted with a hybridization solution which contains the denaturant and at least one oligonucleotide probe.
  • the probe has a nucleotide sequence at least substantially complementary to the nucleotide sequence of the target nucleic acid. Optimization of the hybridization conditions for achieving hybridization of a particular probe to a particular target nucleic acid is well within the level of the person of ordinary skill in the art.
  • fluorescence in situ hybridization first involves fixing the sample to a solid support and preserving the structural integrity of the components contained therein by contacting the sample with a medium containing at least a precipitating agent and/or a cross-linking agent.
  • exemplary agents useful for “fixing” the sample are paraformaldehyde, methanol:acetic acid, and Bouin's solution.
  • Alternative fixatives are well known to those of ordinary skill in the art.
  • the hybridization solution optionally contains one or more of a hybrid stabilizing agent, a buffering agent and a selective membrane pore-forming agent. Following hybridization and subsequent washing, the signal from the fluorescently labeled probe is visualized using imaging techniques well-known to one of ordinary skill in the art.
  • in vitro fertilization or “IVF” is intended to mean fertilization of an egg with sperm outside of a subject. This procedure is often used as a treatment for infertility. Briefly, eggs are retrieved from a subject and mixed with sperm in a culture dish to allow for fertilization. After a period of incubation, usually two or three days, the newly formed embryos are transferred back to the subject. Examples of conditions for which this technique is used include damaged or absent Fallopian tubes, endometriosis, male factor infertility and unexplained infertility.
  • label means a chemical used to facilitate identification and/or quantitation of a target substance.
  • Illustrative labels include fluorescent (e.g., FITC or rhodamine), phosphorescent, chemiluminescent, enzymatic, and radioactive labels, as well as chromophores.
  • label can also refer to a “tag” that can bind specifically to a labeled molecule.
  • the tag can be an epitope or antigen (e.g., digoxigenin), and an enzymatically, fluorescently, or radioactively labeled antibody can be used to bind to the tag for visualization purposes.
  • the probes are peptide nucleic acid (PNA)-labeled probes, and are labeled according to manufacturer's protocols (Applied Biosystems, Inc., Framingham, Mass.).
  • oocyte includes a female germ cell. Oocytes occur in both immature and mature states. “Immature” refers to oocytes that are viable but incapable of fertilization without additional growth or maturation. “Mature” refers to oocytes that have been ovulated from the ova of a female and are capable of being fertilized. Mature also refers to immature oocytes that have been exposed to appropriate hormones or agents to render them capable of fertilization by sperm. Oocytes recovered from “unstimulated” follicles or ovaries are natural oocytes obtained from follicles or ovaries that were not treated with any gonadotropins or other hormones or agents to stimulate maturation of the oocytes.
  • Oocytes recovered from “stimulated” ovaries may be either mature or immature.
  • Subjective criteria to estimate the viability and maturity of the ovum can be done microscopically after removal of the ovum from the follicle, and includes assessing the number and density of surrounding granulosa cells, the presence or absence of the germinal vesicle, and/or the presence or absence of the first polar body.
  • the oocyte is ovulated from a female subject and obtained for use in IVF.
  • the oocyte is immature and is matured in vitro for use in IVF treatment.
  • “Spare” oocytes are those obtained from a female subject for use in IVF procedures but not used for fertilization. Spare oocytes can be obtained from fertile females who are donating their eggs for IVF. In one embodiment of the invention, representative oocytes are chosen from a population of oocytes for the telomere length assay. These representative oocytes may be, for example, spare oocytes.
  • the term “optimizing the viability of the embryo” is intended to mean maximizing the chance of successful embryonic development. Successful development of the embryo is characterized by the embryo being free of congenital defects. In one embodiment of the invention, the viability of the embryo is optimized by selecting oocytes which are unlikely to exhibit aneuploidy.
  • peptide nucleic acid or “PNA” means any oligomer, linked polymer or chimeric oligomer, comprising two or more PNA subunits (residues).
  • peptide nucleic acid-labeled or “PNA-labeled,” used interchangeably herein, refers to a probe labeled with a non-naturally occurring polyamide, which can hybridize to a nucleic acid (DNA or RNA) with sequence specificity (U.S. Pat. No. 5,539,082; Egholm et al. (1993) Nature 365:566-568).
  • PNA-labeled probes examples include the detection of rRNA in ISH and FISH assays (WO95/32305; WO97/18325), the analysis and detection of mRNA, the analysis and detection of viral nucleic acids, and the analysis and detection of centromeric sequences in human chromosomes and human telomeres (Lansdorp et al. (1996) Human Mol. Genetics, 5: 685-691; WO 97/14026).
  • a PNA-labeled probe has also been used to detect human X chromosome specific sequences (WO 97/18325).
  • predisposition to aneuploidy is intended to mean that there is a chance that aneuploidy will occur in a cell because conditions are favorable for chromosomal missegregation.
  • embryos that have a predisposition to aneuploidy are those embryos which develop from an oocyte with telomeres that are abnormal in length.
  • embryos with a predisposition to aneuploidy are those from oocytes that are produced by females that are likely to produce oocytes with abnormally short telomeres.
  • telomeres in the oocytes of a female are shorter than the standard size, resulting in a high probability that the fertilized oocytes will undergo cell cycle arrest shortly after fertilization.
  • reproductive failure indicates a history of recurrent spontaneous abortion, unexplained infertility or implantation failure following in vitro fertilization and embryo transfer.
  • the term also includes, for example, chromosomal abnormalities, depletion of oocytes, embryonic cell cycle arrest, failure of the embryo to implant, and apoptosis Telomerase activity is present during early oogenesis, but is almost absent during late oogenesis and early preimplantation embryo development until the morula stage of development.
  • Telomere length in oocytes and early embryos is established during early development.
  • telomeres reach a critically short length, the cell undergoes cell cycle arrest and apoptosis, so late oogenesis and early preimplantation embryo development may represent a kind of bottleneck for telomere length during development.
  • female patients that have a predisposition for reproductive failure are those female patients with oocytes containing chromosomes with telomeres that are abnormal in length. In another embodiment, female patients that have a predisposition for reproductive failure are identified as having oocytes with abnormally short telomeres.
  • pre-implantation genetic testing is intended to mean testing of a certain genetic locus or loci of a cell which is administered prior to implantation of a fertilized oocyte or oocytes by IVF procedures. In one embodiment of the invention, pre-implantation genetic testing is performed on the polar body of a fertilized or unfertilized oocyte. In another embodiment of the invention, pre-implantation genetic testing is performed on an oocyte or oocytes which are representative of a population of oocytes.
  • probe includes an oligonucleotide designed to hybridize specifically to a target nucleic acid, wherein the hybridization of the probe to the target can be detected.
  • the probe is labeled as described above so that its binding to the target can be visualized.
  • the probe is produced from a source of the target nucleic acid sequence, for example, a collection of clones or a collection of polymerase chain reaction (PCR) products comprising the target sequence, or portion thereof.
  • PCR polymerase chain reaction
  • the source nucleic acid may be processed in some way, for example, by removal of repetitive sequences or blocking them with unlabeled nucleic add with complementary sequence, so that hybridization with the resulting probe produces staining of sufficient contrast on the target.
  • telomere-specific staining of the current invention is accomplished by using nucleic acid probes that hybridize to sequences specific to a telomere target.
  • An example of a telomeric probe of the invention is a FITC-labeled peptide nucleic acid (PNA) probe (Applied Biosystems, Framingham, Mass.) comprising the sequence CCCTAACCCTAACCCTAA (SEQ ID NO: 1).
  • PNA FITC-labeled peptide nucleic acid
  • Other examples of probes that identify telomeres include any one of SEQ ID NOS: 2 through 10.
  • results from the FISH analysis are analyzed using digital fluorescent microscopes to measure the fluorescent signal from probes directed to telomeric DNA sequences. This analysis is then used to quantify the relative length of a telomeres on a chromosome from a cell.
  • the fluorescent signal from the hybridized probe is indicative of the telomere length of the chromosome. For example, the absence of signal would indicate extremely short or absent telomeres on the chromosomes. A low signal would indicate shortened telomeres, while a medium range signal would indicate an average length telomere. Finally, an intense, strong signal would indicate extra long telomeres relative to the average length.
  • standardized average length is intended to mean an average telomere length which is used to determine whether the telomere length of a cell of interest is abnormal.
  • the standardized average length of a cell is determined by measuring the telomere length of a control cell or taking the average telomere length of a population of control cells.
  • oocytes which are known to have fertility are used to determine the standardized average length.
  • one or more oocytes obtained from a woman who has demonstrated fertility and who is known to have oocytes which are not predisposed to aneuploidy can be used to determine the standardized average length of oocytes.
  • spare eggs and polar bodies obtained from young egg donors, whose eggs generate pregnancies can be used to determine the standardized average length.
  • telomere is intended to mean the modified end of an eukaryotic chromosome which contains repeated sequences of DNA.
  • telomeres are composed of many kilobases of simple tandem 5′-TTAGGG repeats (Moyzis et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:6622).
  • the termini of the chromosomes are not fully replicated (Watson (1972) Nature New Biology 239:197,) by the action of DNA polymerase. Incomplete replication occurs at the 3′ end of each of the two template strands of the chromosome, because the RNA primer needed to initiate synthesis in effect masks the 3′ end of the template.
  • RNA primer is degraded after strand synthesis, and, as there are no additional sequences beyond the 3′ end of the template to which primers can anneal, the portion of the template to which the RNA primer hybridized is not replicated. In the absence of other enzymes, the chromosome is thus shortened with every cell division.
  • telomeres consist of a tandem repeat array of a short sequence, which may be desired for experimental manipulation including, e.g., contacting with a probe or primer.
  • human telomeric region and human telomeric repeat sequences are typically referred to herein for illustrative purposes. This illustrative use is not intended to limit the invention, and those of skill in the art will recognize that the present methods can be used to measure telomere length of telomeres from any organism.
  • telomere length is intended to mean the approximate physical measurement of the length of all telomeric repeat sequences at the end of a chromosome relative to a standardized control.
  • the mean telomere length of a population of cells can provide a standardized average length by which to determine if a telomere is long or short.
  • An abnormal telomere length is a telomere length which is greater than or less than the standardized average length of a telomere.
  • telomere length assay is intended to mean a method by which the ends of chromosomes or telomeres of a cell are measured. Measurement of telomere length in cells can predict risk of aneuploidy.
  • the method of the invention uses a telomere length assay to predict the risk of aneuploidy in oocytes in order to minimize the chance of fertilizing oocytes which may give rise to embryos which will be predisposed to cell cycle arrest and embryo death. For example, telomere length of chromosomes from polar bodies (which contain mirror images of chromosomes still in the oocyte) and/or from spare oocytes, provides an estimate of the risk of aneuploidy in resulting embryos.
  • the method of the invention uses a telomere length assay to select oocytes/fertilized oocytes with a low risk of aneuploidy, thereby increasing the likelihood of successful implantation and normal offspring.
  • the telomere length assay is performed using telomeres from chromosomal spreads from unfertilized oocytes and/or polar bodies from fertilized or unfertilized oocytes.
  • telomere length is determined using Q-FISH analysis, wherein PNA-labeled telomere repeat probes are hybridized to telomeric DNA in a cell and analyzed by quantitative FISH using digital microscopy and integrated optical density of the fluorescence signal. Digital imaging technology is readily available in any molecular biology laboratory and most IVF centers.
  • telomere repeats is intended to mean tandem repeats of a specific nucleotide sequence found within the telomeres at the end of chromosomes. In humans and other vertebrates, the telomere repeats are commonly tandem repeats of the sequence TTAGGG. The number of these tandem repeat sequences, present at the ends of a chromosome, determine the telomere length of a chromosome.
  • the invention provides methods for determining the risk of reproductive failure and/or aneuploidy in a cell comprising obtaining at least one chromosome from the cell and measuring the telomere length of the chromosome to thereby determine the risk of aneuploidy in the cell.
  • cells include an oocyte, an oocyte representative of a population of oocytes, a polar body form a fertilized oocyte or a polar body from an unfertilized oocyte.
  • the telomere is preferably detected by a labeled telomere-specific probe which is hybridized to the chromosome prior to measuring telomere length of the chromosome.
  • the probe is hybridized to telomere repeats.
  • the probe is peptide nucleic acid (PNA)-labeled, preferably FITC-labeled (CCCTAA) 3 (SEQ ID NO: 10).
  • the telomere is advantageously measured using quantitative fluorescent in situ hybridization (Q-FISH) analysis, although any other measuring methods know in the art may be used.
  • Q-FISH quantitative fluorescent in situ hybridization
  • the oocyte selected is representative of a population of oocytes and a probe is obtained for hybridizing to the chromosome of the oocyte.
  • the probe is a labeled telomere-specific probe comprising a nucleic acid sequence identified by any one of SEQ ID NOS: 1-10.
  • the nucleic acid sequence comprises a sequence having at least about 80 percent sequence identity to any one of SEQ ID. NOS. 1 through 10; more preferably, the nucleic acid sequence comprises a sequence having at least about 90 percent sequence identity to any one of SEQ ID. NOS. 1 through 10; still more preferably the nucleic acid sequence comprises a sequence having at least about 100 percent sequence identity to any one of SEQ ID. NOS. 1 through 10.
  • the invention provides assays to determine the predisposition of cells, for example, an oocyte to reproductive failure and/or aneuploidy in order to optimize the implantation and viability of the embryo.
  • the invention provides a method of pre-implantation genetic testing for screening oocytes with a predisposition to aneuploidy. Oocytes which have a predisposition to aneuploidy are more likely to give rise to embryos with developmental defects. The oocytes are screened for use in in vitro fertilization. To optimize the overall success of in vitro fertilization and to maximize the chance of a normal embryo, oocytes with abnormally long or short telomeres are not fertilized and/or implanted in the patient undergoing IVF treatment.
  • telomere lengths are determined by Q-FISH analysis, using PNA-labeled probe, quantitative digital microscopy, and integrated optical density of the fluorescence signal.
  • the invention features a method for distinguishing oocytes which have a predisposition to reproductive failure and/or aneuploidy in order to optimize the viability of an embryo for in vitro fertilization treatment.
  • chromosomes are obtained from the oocytes and/or polar bodies to analyze the length of their telomeres.
  • an oocyte is selected for the telomere length assay from a population of oocytes.
  • the telomere length of the selected oocyte is representative of the population of oocytes.
  • the representative oocyte can be a spare oocyte which is not intended for use in IVF.
  • a polar body from a fertilized or unfertilized oocyte is obtained, and its chromosomes are used in the telomere length assay of the invention.
  • Polar bodies contain mirror images of chromosomes from the oocyte from which they are obtained.
  • Polar bodies and oocytes are obtained through techniques known to those of ordinary skill in the art of in vitro fertilization procedures.
  • telomeric DNA from oocytes and/or polar bodies can be used to predict the developmental potential of an embryo based on the correlation between telomere length and a predisposition of the oocyte or polar body to aneuploidy.
  • Standard molecular biology techniques known to one of ordinary skill in the art can be used to determine the length of a telomere, including, for example, quantitative polymerase chain reaction (PCR) (Cawthon (2002) Nucleic Acids Research, 30 : 1 -6).
  • In situ techniques can also be used to measure telomere length, including, for example, primed in situ labeling (PRINS) and fluorescent in situ hybridization (FISH) (Therkelsen, et al.
  • PRINS primed in situ labeling
  • FISH fluorescent in situ hybridization
  • Q-FISH quantitative FISH
  • a female's predisposition to reproductive failure is determined by the length of the telomeric ends of a chromosome.
  • An oocyte is selected for the telomere length assay from a population of oocytes.
  • the telomere length of the selected oocyte is representative of the population of oocytes.
  • the representative oocyte can be a spare oocyte which is not intended for use in IVF.
  • a polar body from a fertilized or unfertilized oocyte is obtained, and its chromosomes are used in the telomere length assay of the invention.
  • telomere length is measured using Q-FISH analysis, which is described below.
  • chromosomes are obtained from a cell or cells of interest, and are morphologically preserved.
  • the chromosome sample, or chromosome spread is prepared according to standard techniques such that individual chromosomes remain substantially intact and typically comprise metaphase spreads or interphase nuclei.
  • Cells described in the methods of the invention include polar bodies and oocytes. Cells can be morphologically preserved by fixation using conventional methods (see for example, A. G. Everson Pearce (1980) Histochemistry Theoretical Applied, 4th Ed., Churchill Livingstone, Edinburgh, for details relating to the general techniques for preparing and fixing tissue).
  • the cells of the invention can be fixed on a support, such as slides or filters.
  • a support such as slides or filters.
  • oocytes can be fixed by spinning small volumes of cells onto slides.
  • Chromosomal preparations or the chromosome spread can also be pre-treated on the support. Pretreatments include, but are not limited to, RNAse treatment to remove endogenous RNA, protease treatment to increase the accessibility by digesting protein surrounding the telomeres, and detergent treatment when it is suspected that lipid membrane components have not been extracted by other procedures.
  • the chromosome spread is then treated with a telomere-specific probe.
  • the probe is a nucleic acid, or analog thereof, which is capable of hybridizing to telomeric DNA. Nucleic acid analogs differ from natural DNA in that they do not have a deoxyribose or ribose backbone.
  • probes are constructed from the nucleic acid analog called peptide nucleic acid (PNA) which contains a polyamide backbone (described in WO 92/2070). PNA probes have been used in FISH to study telomeres in hematopoietic cells (Lansdorp et al.
  • telomere sequences include analogs having cyclic backbone moieties comprising furan or morpholine rings, or acyclic backbone moieties (WO 86/05518). Determination of whether a probe hybridizes to a telomeric sequence can be accomplished by hybridizing the probe to a nucleic acid molecule comprising multiple copies of the telomeric repeat sequences using the hybridization media and conditions described herein.
  • the probe for detecting and quantitating the length of a telomere in a chromosome is a PNA-labeled probe having the following sequence: CCCTAACCCTAACCCTAA (SEQ ID NO: 1).
  • the probe for measuring the length of a telomere contains the repeat sequence TTAGGG (SEQ ID NO: 2), CCCTAA (SEQ ID NO: 3), CCCCAA (SEQ ID NO: 4), CCCCAAAA (SEQ ID NO: 5), CCCACA (SEQ ID NO: 6), CCCTAAA (SEQ ID NO: 7), CCCCT (SEQ ID NO: 8), CCCTAA (SEQ ID NO: 9), or FITC-labeled (CCCTAA) 3 (SEQ ID NO: 10).
  • the telomere specific probe comprises a nucleic acid sequence having at least about 80 percent sequence identity to any one of SEQ ID. NOS. 1 through 10; more preferably, at least about 90 percent sequence identity to any one of SEQ ID. NOS. 1 through 10; still more preferably, at least about 100 percent sequence identity to any one of SEQ ID. NOS. 1 through 10.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • nucleic acid “identity” is equivalent to nucleic acid “homology”.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at online through the Genetics Computer Group), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • GAP program in the GCG software package (available at online through the Genetics Computer Group), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • parameters to be used in conjunction with the GAP program include a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Meyers and Miller ( Comput. Appl. Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or version 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the probe used in the method of the invention is preferably labeled with one or more detectable substances.
  • the probe of the invention is labeled with a detectable substance so that formed hybrids can be visualized microscopically after the in situ hybridization procedure. Visualization can be achieved either directly or indirectly depending on the nature of the label used for the probe.
  • Detectable substances which can be used in direct visualization methods of the probe include, but are not limited to, fluorophores, isotopes, and chemiluminescent compounds. Examples of isotopes include iodine I 125 , I 131 or tritium.
  • FITC fluorescein-isothiocyanate
  • TRITC tetramethyl rhodamine isothiocyanate
  • AMCA amino-methyl coumarin acetic acid
  • Cy2, Cy3, or Cy5 Biological Detection Systems, Pittsburgh, Pa.
  • An example of a chemiluminescent material is luminol.
  • the probe of the invention can be labeled with any of these detectable substances using methods conventionally known in the art.
  • the probe can also be labeled with a detectable substance which is detected in an indirect method.
  • the probe can be labeled with a detectable substance so that formed hybrids are visualized after reacting with an element (e.g., a substrate, an antibody, etc.) which results in a detectable signal.
  • Detectable substances which may be used in indirect methods include enzymes (e.g., horseradish peroxidase, alkaline phophatase, ⁇ -galactosidase, or acetylcholinesterase) and haptens (e.g., biotin, digoxigenin).
  • in situ hybridized sequences are detected using (strept)avidin or anti-biotin antibodies.
  • the probe may be labeled with enzymes and haptens using conventional methods known in the art, for example, in Pattersen et al. (1993) Science, 260:976-979.
  • the probe of the invention can also be labeled with more than one detectable substance.
  • the probe may be labeled with two or more fluorophores.
  • the probe may also be labeled with substances detectable for both direct and indirect detection methods.
  • the method of the invention can also be practiced using differently labeled probes. For example, the method may use three probes having the same nucleic acid sequence but each labeled with a different detectable substance, such as three different fluorophores.
  • the method of the invention is performed using a probe and a counterstain.
  • a counterstain can be used to visualize another feature of the cell (e.g., nucleic acids, cell walls, nuclei, etc.), and includes, for example, DAPI and Hoechst stains.
  • a counterstain can be used to visualize the complete chromosome to compare with the visual signal from the telomere-specific probe.
  • the detectable substance of the probe may provide a colorimetric, photometric, radiometric, etc. signal which can be detected by a wide variety of means.
  • the method of the invention could employ a suitable detectable device capable of detecting a change in absorption density, a change in fluorescence, or radioactive emission, or a shift in the absorbance of a characteristic ⁇ max , for example, the ⁇ max of the detectable molecule, as detailed in the Examples which follow.
  • the probe is labeled with a fluorophore such as fluorescein-isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC), amino-methyl coumarin acetic acid (AMCA) (Molecular Probes, Eugene, Oreg.), Texas Red (Molecular Probes, Eugene, Oreg.), or carboxylmethylindocyano dyes such as Cy2, Cy3, or Cy5 (Biological Detection Systems, Pittsburgh, Pa.).
  • FITC fluorescein-isothiocyanate
  • TRITC tetramethyl rhodamine isothiocyanate
  • AMCA amino-methyl coumarin acetic acid
  • Cy2 Cy3, or Cy5 Biological Detection Systems, Pittsburgh, Pa.
  • Images of fluorophore labeled probes hybridized to target telomeric nucleic acid sequences can be created using conventional techniques known in the art (see also Nederlof, et al (1992) Cytometry 13:839-845).
  • the images are formed electronically, and most preferably are digital (discussed further below).
  • hybridization medium has a low ionic strength and typically contains a buffer, denaturing agent and a blocking reagent.
  • buffers include TRIS and HEPES.
  • suitable denaturing agents include formamide and DMSO.
  • a blocking reagent is any reagent which substantially blocks non-specific binding of the probe.
  • the hybridization medium comprising the telomeric-specific probe is applied to the chromosomal DNA.
  • the hybridization probe and the chromosomal DNA are denatured simultaneously by heat or pH treatment.
  • the probe and telomeric DNA are hybridized under stringent conditions.
  • the image of the hybridized to a detectable probe is captured using imagery equipment standard in a molecular biology or IVF laboratory.
  • instrumentation needed to create a digital image consists of a microscope system, a detector for collection of images, and computer hardware and software for image analysis.
  • fluorescence microscopy is used to quantitatively analyze the FISH results.
  • objective lenses are generally used with high magnification and high numerical aperture, since these characteristics determine the spatial resolution and light collecting properties.
  • a Leitz Dialux epifluorescence microscope equipped with a 100 W mercury-arc lamp and a Neofluor 1,40 NA oil objective can be used to measure fluorescence, and the filter used for selection of the fluorophore may be as follows: PL450-SP490 (excitation filter), DM510 (dichroic mirror), and LP515-SP560 (emission filters).
  • a detector for imaging the FISH results should be selected so that it has sufficient sensitivity to detect the fluorescing signals obtained by FISH, for example, detection of fluorescing signals that can be differentiated from background fluorescence, as detailed in the Examples which follow.
  • the detector also provides a linear response to a wide range of wavelengths, has a high signal to noise ratio, and has wide dynamic range and little geometric aberrations.
  • the intensity of a spot may be calculated as the integrated intensity of all pixels within the area of the spot corrected for the background.
  • the length of the telomere is quantitated from the specific fluorescent intensity of all the pixels within the area of the spot (telomeric region) corrected for the background.
  • telomere fluorescence In general, computer image analysis is used to optimize signal to noise ratio of telomere fluorescence from human oocytes and polar bodies. Telomeres can be imaged using a number of commercially available computer image analysis programs. An example of a commercially available analysis program is the MetaMorph® Imaging System program (Universal Imaging Corporation (Downingtown, Pa.). Software, which can be programmed to integrate intensity and area of telomere fluorescence, is also available in the public domain; e.g., NIH provides image analysis software at no cost. It will be appreciated that macro programs can also be written for commercially available software, which will further optimize the signal to noise ratio of telomere fluorescence from human oocytes and polar bodies.
  • telomere length Calibration of the Q-FISH technique for telomere length is performed using known telomere lengths, including, for example, genetically engineered artificial telomeres. Genetically engineered artificial telomeres can be generated based on the determined standardized average length. In another example, chromosome spreads from mouse oocytes from strains known to have long and/or short telomeres can also be used to calibrate telomere length. Examples of mouse strains known to have abnormal length telomeres include the TR ⁇ / ⁇ strain (Blasco, et al. (1997) Cell 91:25-34) and the ATM ⁇ / ⁇ strain (Hande et al. (2001) Hum. Mol. Genet. 10:519-528).
  • Telomeres are quantified as being average, longer, or shorter than the determined standard telomere length of the cell whose telomeres were analyzed.
  • the standard telomere length of a cell can be determined by the results obtained from Q-FISH analysis of a control telomere. Measurement of telomere length is further described in Martens et al. (1998) Nature Genetics 18:76-80. Cells whose chromosomes are determined to have abnormal length telomeres are likely to give rise to aneuploidy.
  • an oocyte which is analyzed by Q-FISH and is determined to have abnormal length telomeres is not used for in vitro due to a predisposition to aneuploidy.
  • a polar body is analyzed by Q-FISH analysis for a predisposition to aneuploidy.
  • GV oocytes were matured in vitro for 24 to 48 hours until they reached MII stage of development.
  • Q-FISH has become the method of choice for examination of both telomere length and loss in single cells (Zijlmans, Martens et al. 1997 , PNAS USA, 94: 7423-7428.).
  • Chromosome spreads were prepared by a hypotonic treatment of oocytes or spermatocytes with 1% sodium citrate for 20 min, followed by fixation in methanol:acetic acid (3:1), and air dried.
  • Chromosomes were counter-stained with 0.2 ⁇ g/ml Hoechst 33342. Embryos were mounted onto a glass slide in Vectashield mounting medium (Vector Laboratories, Burlingame, Calif.). Telomeres were detected with a FITC filter using a Zeiss fluorescence microscope (Axioplan 2 imaging) and images were captured by an AxioCam using AxioVision 3.0 software.
  • Q-FISH Quantitative fluorescence in situ hybridization
  • telomere fluorescence intensity was integrated using the TFL-TELO program (Poon, Martens et al. 1999 , Cytometry, 36: 267-278), kindly provided by P. Lansdorp, and calibrated with fluorescence beads. Telomere length (kb) was estimated based on the relative fluorescence intensity, using standard cell lines with known telomere length (McIlrath, Bouffler et al. 2001 , Cancer Res., 61(3): 912-5).
  • telomere length is determined from spare oocytes and polar bodies of control oocytes. Telomere lengths are determined by the number of tandem repeats of a specific nucleotide sequence found within the telomeres at the end of chromosomes. In humans and other vertebrates, the telomere repeats are commonly tandem repeats of the sequence TTAGGG.
  • Control oocytes and/or polar bodies are obtained from donors undergoing IVF to donate their oocytes to women with egg infertility.
  • oocytes can also be retrieved for control studies from women undergoing intracytoplasmic sperm injection (ICSI) for severe male factor infertility or egg donation.
  • ICSI intracytoplasmic sperm injection
  • Other factors considered for women donors include having a day three FSH values less than 9 IU/ml, inhibin B levels greater than 45 and estradiol levels less than 80 ⁇ g/ml, at least 12 eggs retrieved after COH with standard gonadotropin dosing, and no known diagnosis of female infertility.
  • Oocytes are retrieved by transvaginal needle aspiration from the ovary, performed under gentle intravenous sedation after controlled ovarian hyperstimulation (COH), per standard protocols. Methods for obtaining eggs for egg donation are described in Klein, et al. (2002) Best Pract. Res. Clin. Obstet. Gynaecol. 16:277-291.
  • Human oocytes are cultured according to standard, published clinical protocols. Protocols for culturing human oocytes can also be found throughout the literature, including, for example, Quinn et al. (1998) Fertil. Steril. 69:399-402.
  • Polar bodies from control oocytes are obtained and used as a control according to whether the oocyte from which they arose went on to form normal babies after the resulting embryos are transferred into the uterus of the recipient women.
  • Polar bodies are biopsied according to standard protocols, as described, for example, in Verlinsky et al. (1996) J. Assist. Reprod. Genet. 13(2): 157-162. Briefly, for biopsy of polar bodies, first the egg or oocyte was placed on the holding pipette of a micromanipulator under 40 X objective on an inverted microscope. The zona was broached by laser, acid tyrodes, pronase or mechanical dissection.
  • the polar body was teased out with gentle suction into a biopsy pipette affixed to the second micromanipulator. The polar body was then released onto a slide where a chromosome spread was made. The first polar body was obtained before fertilization, while the second was obtained after.
  • oocytes are also obtained from women who are known to have dysfunctional oocytes. Eggs are retrieved by transvaginal aspiration under light intravenous sedation after controlled ovarian hyperstimulation (COH). Dysfunctional eggs are retrieved from women undergoing ICSI for mild male factor and/or egg factor infertility, who have day three FSH values greater than 14 IU/ml, inhibin B levels less than 16 and estradiol levels greater than 80 ⁇ g/ml, five or fewer eggs retrieved after COH with high dose (at least FSH 450 IU/day) gonadotropin dosing, and known diagnosis of female infertility.
  • COH controlled ovarian hyperstimulation
  • telomere length of control oocytes and polar bodies is quantitatively analyzed through Q-FISH analysis, as described in the materials and methods section and example III.
  • Q-FISH Quantitative Fluorescence In situ Hybridization
  • Q-FISH analysis is used to determine the telomere length of chromosomes from oocytes.
  • Oocytes are obtained from a patient who is interested in determining the risk of aneuploidy in her oocytes. From the batch of oocytes obtained from the ova, a spare oocyte or oocytes are taken for Q-FISH analysis. Results obtained for spare oocytes are representative of the population of oocytes in the batch.
  • Control oocytes are obtained from a woman with demonstrated fertility and are prepared according to the methods described in the materials and methods section and Example I. Control oocytes are analyzed prior to Q-FISH analysis of the oocyte of interest or, alternatively, are prepared for Q-FISH analysis alongside the experimental spare oocyte(s).
  • telomere length from control oocytes is the standardized control to which the spare oocytes are compared.
  • Chromosomal spreads are prepared by hypotonic treatment of oocytes with 1% sodium citrate for 20 min, followed by fixation in methanol:acetic acid (3:1). Chromosomal spreads are then allowed to air dry.
  • Fluorescence in situ hybridization is performed according to the manufacturer's protocol with FITC-labeled peptide nucleic acid (PNA) probe (Applied Biosystems, Framingham, Mass.) comprising the sequence CCCTAACCCTAACCCTAA (SEQ ID NO: 1).
  • PNA FITC-labeled peptide nucleic acid
  • the telomere specific probe is hybridized under stringent conditions to the chromosomal spreads. Chromosomes are then counterstained with Hoechst 33342 at a concentration of 0.2 ⁇ g/ml. Chromosomes are mounted onto a glass slide in Vectashield mounting medium (Vector Laboratories, Burlingame, Calif.), and analyzed using fluorescence microscopy.
  • Telomere length is determined by quantitative digital microscopy and integrated optical density of the fluorescence signal.
  • the signal from the FITC-labeled probe hybridized to the telomeres is detected using a Zeiss fluorescence microscope (Axiophot) with a FITC filter. Images of fluorescing telomeres are captured using an AxioCam digital microscope camera using AxioVision 2.0 software. Commercially available software, including MetaMorph (Universal Imaging CorporationTM, Downingtown Pa.), is used to integrate the fluorescent intensity and area. The digital images are then used for quantitative analysis of telomere length based on criteria described in Zijlmans et al. (1997).
  • telomere Fluorescence Units or “TFUs” and plotted on a graph for comparative purposes. Determined telomere lengths of the spare oocytes are compared to the results obtained for the control oocytes to establish whether the spare oocyte telomere lengths are abnormal.
  • telomere lengths are determined by the number of tandem repeats of a specific nucleotide sequence found within the telomeres at the end of chromosomes. In humans and other vertebrates, the telomere repeats are commonly tandem repeats of the sequence TTAGGG.
  • telomere length by Q-FISH analysis can be repeated for a polar body or polar bodies which are obtained from fertilized or unfertilized oocytes.
  • Polar bodies are obtained through conventional methods known to one of ordinary skill in the art.
  • telomere maximum (19.3 ⁇ 3.1 k.b. vs. 13.9 ⁇ 3.28 k.b., p ⁇ 0.01) and mean (7.5 ⁇ 1.17 k.b. vs. 6.2 ⁇ 1.69 k.b.) lengths were significantly longer and standard deviation greater (4.4 ⁇ 0.96 vs. 3.5 ⁇ 1.12) in eggs from patients who went on to become pregnant, compared to those who failed attempts at pregnancy.
  • Minimum telomere length and number of missing telomere signals did not differ between groups.
  • telomere length predicts favorable reproductive outcome in infertile women undergoing IVF. Telomere length provided a better predictor of pregnancy outcome following IVF than patient age itself or other clinical parameters, including when telomere length was measured only in spare eggs.
  • Q-FISH analysis is performed, as described above in the materials and methods section and Example III, to determine the length of the oocyte's telomeres.
  • the length of telomeres within the population is assumed to be similar, therefore allowing the ordinarily skilled artisan to determine whether the population of oocytes might be at risk for aneuploidy.
  • telomere length of the polar body. Determining the telomere length allows the ordinarily skilled artisan to assess the risk of aneuploidy, and subsequently whether the oocyte is likely to give rise to an abnormal embryo.
  • telomeric FISH was performed on oocyte metaphase spreads using a fluorescent FITC-labeled (CCCTAA) 3 (SEQ ID NO: 10) peptide nucleic acid (PNA) probe, which is able to detect 200 bp of TTAGGG repeats at the telomeres.
  • FISH was employed to measure telomere length in chromosomes spread from spare eggs matured from the GV stage after aspiration from consenting subjects undergoing ART and ICSI ( FIG. 1 ).
  • telomere lengths were normally distributed. Although clinical characteristics did not distinguish the pregnant vs. the non-pregnant group, nearly all measures of telomere length did differ significantly between the pregnant and non-pregnant groups (t-test) (Table 2). Mean and maximum telomere lengths were greater in oocytes from women who became pregnant compared to those who did not become pregnant. Variation in telomere length, measured by standard deviation, also was significantly greater in oocytes from women who became pregnant compared to those who did not (F test). No women became pregnant in this study who had a mean telomere length in any spare oocytes less than 6.32 k.b.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Surgery (AREA)
  • Pathology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Transplantation (AREA)
  • Biomedical Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Medical Informatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US10/531,964 2002-10-16 2005-12-07 Methods of assessing the risk of reproductive failure by measuring telomere length Abandoned US20060141461A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/531,964 US20060141461A1 (en) 2002-10-16 2005-12-07 Methods of assessing the risk of reproductive failure by measuring telomere length
US12/482,176 US8771941B2 (en) 2002-10-16 2009-06-10 Methods of assessing the risk of reproductive failure by measuring telomere length
US14/323,638 US20150167081A1 (en) 2002-10-16 2014-07-03 Methods of assessing the risk of reproductive failure by measuring telomere length

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US41907102P 2002-10-16 2002-10-16
US45274103P 2003-03-07 2003-03-07
PCT/US2003/032672 WO2004035597A2 (fr) 2002-10-16 2003-10-13 Procede permettant d'evaluer le risque d'echec de la reproduction par mesure de la longueur d'un telomere
US10/531,964 US20060141461A1 (en) 2002-10-16 2005-12-07 Methods of assessing the risk of reproductive failure by measuring telomere length

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/032672 A-371-Of-International WO2004035597A2 (fr) 2002-10-16 2003-10-13 Procede permettant d'evaluer le risque d'echec de la reproduction par mesure de la longueur d'un telomere

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/482,176 Continuation US8771941B2 (en) 2002-10-16 2009-06-10 Methods of assessing the risk of reproductive failure by measuring telomere length

Publications (1)

Publication Number Publication Date
US20060141461A1 true US20060141461A1 (en) 2006-06-29

Family

ID=32110212

Family Applications (3)

Application Number Title Priority Date Filing Date
US10/531,964 Abandoned US20060141461A1 (en) 2002-10-16 2005-12-07 Methods of assessing the risk of reproductive failure by measuring telomere length
US12/482,176 Expired - Fee Related US8771941B2 (en) 2002-10-16 2009-06-10 Methods of assessing the risk of reproductive failure by measuring telomere length
US14/323,638 Abandoned US20150167081A1 (en) 2002-10-16 2014-07-03 Methods of assessing the risk of reproductive failure by measuring telomere length

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/482,176 Expired - Fee Related US8771941B2 (en) 2002-10-16 2009-06-10 Methods of assessing the risk of reproductive failure by measuring telomere length
US14/323,638 Abandoned US20150167081A1 (en) 2002-10-16 2014-07-03 Methods of assessing the risk of reproductive failure by measuring telomere length

Country Status (3)

Country Link
US (3) US20060141461A1 (fr)
AU (1) AU2003284234A1 (fr)
WO (1) WO2004035597A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014152676A1 (fr) * 2013-03-15 2014-09-25 New York University Procédé pour l'analyse sur une cellule individuelle de longueur de télomère
WO2015179301A1 (fr) * 2014-05-19 2015-11-26 Eleftherios Papoutsakis Particules et microparticules mégacaryocytaires pour la thérapie cellulaire & la modification du destin de cellules souches et progénitrices

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008081451A2 (fr) * 2007-01-03 2008-07-10 Monaliza Medical Ltd. Méthode et trousse d'analyse du matériel génétique d'un foetus
US8084203B2 (en) 2008-05-12 2011-12-27 Fundacion Centro Nacional De Investigaciones Oncologicas Carlos Iii Methods for the determination of telomere length in a semi-automatic manner of every single cell in a immobilized cell population
CA2723950C (fr) * 2008-05-12 2016-07-12 Fundacion Centro Nacional De Investigaciones Oncologicas Carlos Iii Procedes et reactifs pour la determination de la longueur de telomeres d'une maniere semi-automatique de chaque cellule individuelle dans une population de cellules immobilisees
WO2010124101A2 (fr) 2009-04-22 2010-10-28 Juneau Biosciences, Llc Marqueurs génétiques associés à l'endométriose et utilisation de ceux-ci
US9177098B2 (en) 2012-10-17 2015-11-03 Celmatix Inc. Systems and methods for determining the probability of a pregnancy at a selected point in time
WO2018187585A1 (fr) * 2017-04-06 2018-10-11 Celmatix Inc. Procédés pour l'évaluation du potentiel de succès de reproduction et d'information de traitement à partir de tels procédés
EP3674418A1 (fr) * 2018-12-26 2020-07-01 Life Length S.L. Procédé de mesure de variables associés à un télomère et leurs utilisations pour le diagnostic et/ou le pronostic de maladies associées à un télomère
EP4581367A1 (fr) * 2022-09-01 2025-07-09 The Johns Hopkins University Procédés de mesure de longueur de télomère

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5741677A (en) * 1995-06-07 1998-04-21 Geron Corporation Methods for measuring telomere length

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NO20014865D0 (no) 2001-10-05 2001-10-05 Ericsson Telefon Ab L M Optimalisering av handover-prosedyrer i GPRS

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5741677A (en) * 1995-06-07 1998-04-21 Geron Corporation Methods for measuring telomere length

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014152676A1 (fr) * 2013-03-15 2014-09-25 New York University Procédé pour l'analyse sur une cellule individuelle de longueur de télomère
WO2015179301A1 (fr) * 2014-05-19 2015-11-26 Eleftherios Papoutsakis Particules et microparticules mégacaryocytaires pour la thérapie cellulaire & la modification du destin de cellules souches et progénitrices
US10538738B2 (en) 2014-05-19 2020-01-21 Eleftherios Papoutsakis Megakaryocytic particles and microparticles for cell therapy and fate modification of stem and progenitor cells
US11820968B2 (en) 2014-05-19 2023-11-21 Eleftherios Papoutsakis Megakaryocytic particles and microparticles for in vivo hematopoietic cell and gene therapies

Also Published As

Publication number Publication date
WO2004035597A3 (fr) 2004-09-16
WO2004035597A2 (fr) 2004-04-29
US20090326316A1 (en) 2009-12-31
US20150167081A1 (en) 2015-06-18
AU2003284234A1 (en) 2004-05-04
US8771941B2 (en) 2014-07-08
AU2003284234A8 (en) 2004-05-04

Similar Documents

Publication Publication Date Title
US8771941B2 (en) Methods of assessing the risk of reproductive failure by measuring telomere length
Wilton Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization
US11149314B2 (en) Methods for determining the quality of an embryo
US20080085836A1 (en) Method for genetic testing of human embryos for chromosome abnormalities, segregating genetic disorders with or without a known mutation and mitochondrial disorders following in vitro fertilization (IVF), embryo culture and embryo biopsy
Pujol et al. Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies
EP2450456A2 (fr) Evaluation de la compétence d'oocyte
Frumkin et al. Elucidating the origin of chromosomal aberrations in IVF embryos by preimplantation genetic analysis
Azmanov et al. Profile of chromosomal aberrations in different gestational age spontaneous abortions detected by comparative genomic hybridization
US6979541B1 (en) Methods for identifying chromosomal aneuploidy
Keskintepe et al. Reproductive oocyte/embryo genetic analysis: comparison between fluorescence in-situ hybridization and comparative genomic hybridization
AU2005252470B2 (en) Identifying chromosomal abnormalities in cells obtained from follicular fluid
Vanderwal Preimplantation genetic screening: Changes in technology and methodology, ethical implications, and areas of future research
Martorell et al. Chromosome 16 abnormalities in embryos and in sperm from a male with a fragile site at 16q22. 1
Jeremić et al. Preimplantation genetic testing.
Lewin et al. Preimplantation genetic diagnosis for infertility
Forman et al. The genetics of pregnancy failure
Baart et al. Chromosomes Development: Abnormalities, in Incidence Underlying Early Human of Mechanisms, Chromosomal
Fragouli et al. Genomics for Embryo Selection
CZ202462A3 (cs) Metoda rozlišení meiotických a mitotických vad při preimplantačním a prenatálním vyšetření
McArthur et al. Embryo Biopsy for PGD: Current Perspective
Hultén et al. Errors in chromosome segregation during oogenesis and early embryogenesis
Wilton et al. Use of comparative genomic hybridisation (CGH) and microarray-CGH for preimplantation genetic screening
Fragouli et al. The origins of aneuploidy in human embryos
Class et al. Patent application title: METHODS FOR DETERMINING THE QUALITY OF AN EMBRYO Inventors: Samir Hamamah (Montpellier, FR) Safia El Messaoudi (Montpellier, FR) Alain Thierry (Montpellier, FR) Said Assou (Montpellier, FR)
Zagradišnik et al. Detection of aneuploidy using multiplex ligation–dependent probe amplification in fetal tissues from aborted pregnancies: Določanje anevploidij z metodo pomnoževanja od ligacije odvisnih prob v fetalnih tkivih splavkov

Legal Events

Date Code Title Description
AS Assignment

Owner name: WOMEN AND INFANTS HOSPITAL OF RI, RHODE ISLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KEEFE, DAVID;REEL/FRAME:016964/0710

Effective date: 20051027

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: KEEFE, DAVID L., RHODE ISLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:WOMEN AND INFANTS HOSPITAL OF RHODE ISLAND, INC.;REEL/FRAME:022884/0979

Effective date: 20090622